Solubilization and functional reconstitution of the branched-chain alpha-keto acid transporter from rat heart mitochondria.
The mitochondrial branched-chain alpha-keto acid transporter was solubilized from rat heart mitochondria and its function was reconstituted in phospholipid vesicles. The transporter was extracted from mitoplasts with Triton X-114 in the presence of exogenous cardiolipin and alpha-ketoisocaproate. Upon incorporation of this extract into asolectin vesicles by the freeze-thaw-sonication technique, a p-chloromercuribenzoate-sensitive, protein-dependent transport of alpha-ketoisocaproate into the proteoliposomes was observed. Significant inhibition of alpha-ketoisocaproate transport was observed in the reconstituted system with branched-chain alpha-keto acids (64-83%) and the related carboxylates alpha-ketocaproate (58%) and alpha-ketovalerate (49%), but not with substrates for the pyruvate carrier (less than 5%). The reconstituted carrier was substantially inhibited by sulfhydryl reagents, by the histidine-specific reagent diethyl pyrocarbonate, and by the tyrosine-specific reagent N-acetylimidazole. The extraction and functional reconstitution of the branched-chain alpha-keto acid transporter represents an important first step towards purification and molecular characterization of this anion carrier.
Hutson
S M
SM
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27103.
Roten
S
S
Kaplan
R S
RS
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D007651:Keto Acids / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008929:Mitochondria, Heart / Q000378:metabolism*; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013367:Submitochondrial Particles / Q000378:metabolism; D013379:Substrate Specificity
D002352:Carrier Proteins; D007651:Keto Acids; D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; C020485:proteoliposomes; C024990:asolectin